Our Broad Challenge is to develop enabling technologies for the prevention and treatment of diseases affecting the bladder. The Proof of Concept that we are testing is that intravesical instillation of small interference RNA (siRNA) packaged in biodegradable nanospheres provides wide opportunities for the treatment of urologic diseases including transitional cell carcinoma of the bladder (TCC), interstitial cystitis (IC), overactive bladder, and detrusor hyperreflexia. Because of their robust, gene selective silencing of target protein expression, siRNA oligonucleotides are an attractive therapeutic option with high selectivity and specificity and minimal toxicity to neighboring cells. siRNAs, however, have a relatively short half-life and thus we will address the technical challenges of stabilization and intravesical delivery of siRNAs. To achieve this goal, we plan to create and test clinically viable, non-viral nanosphere siRNA complexes that are intravesically instilled for treatment of diseases of the urinary tract. This past year, in collaboration with Drs. W. Mark Saltzman and Kim Woodrow of the Yale University Department of Bioengineering, we have shown that antennapedia (AP), a Drosophila transcription factor that facilitates uptake of peptides and oligonucleotides into mammalian cells, when complexed with PLGA nanoparticles (AP-PLGA) adheres effectively to T-24 bladder cancer cells, and slowly releases complexed siRNA over 10 days in amounts sufficient to downregulate intracellular target mRNA and protein. Conversely, naked survivin and VEGF siRNA are stable for only a few hours. We have complexed two siRNAs with AP-PLGA, survivin siRNA and VEGF siRNA, both with relevance in urological diseases. The Inhibitor of Apoptosis (IAP), survivin, is not detected in normal urothelium and its expression in bladder cancer correlates with poor prognosis. The angiogenic factor, VEGF is overexpressed in TCC and may play an important role in the pathogenesis of TCC and interstitial cystitis. We have shown that these survivin siRNA-AP-PLGA and VEGF siRNA-AP-PLGA are released into the T-24 cells and down-regulate targeted mRNA and protein. Furthermore, VEGF siRNA-AP-PLGA downregulates VEGF levels in normal human urothelium. We now plan to: 1) develop and test a number of nanoparticle controlled release systems containing targeting proteins that will stabilize and deliver siRNAs and drugs, to test their physicochemical properties (materials testing), and the capacity of the nanoparticles to release siRNA efficiently from the nanospheres to an intracellular target site;2) move from an in vitro to an in vivo system and test in a whole animal the ability of human targeted encapsulated siRNAs to reduce target protein and mRNA and cell/tumor growth;and 3) test whether combinations of encapsulated siRNAs and/or histone deacetylase inhibitors (HDACIs) more effectively reduce tumor burden, the time to onset, rate of occurrence and mortality compared to individual encapsulated siRNAs in a nitrosamine induced bladder cancer model. The ultimate challenge is to design more effective intravesical instillation protocols for treatment of common urological diseases including bladder cancer, overactive bladder and IC using siRNAs encapsulated in microspheres to increase their stability and prolong their efficacy. Standard chemotherapeutic treatment options for urological diseases including bladder cancer, overactive bladder and interstitial cystitis may cause undesirable side effects or may be ineffective. Small interference RNA (siRNA) can specifically and sensitively degrade RNA messages and thus reduce levels of the proteins synthesized from the specific mRNAs that may play a role in disease development. In order to exploit the therapeutic potential of these siRNAs, which are short lived and easily degraded, we have designed strategies to stabilize and test them. We plan to encapsulate the siRNAs in polymer nanoparticles which will release the siRNA over days to weeks. We also will add peptides that will target the nanoparticles to specific cells within the bladder. These siRNA polymers will be instilled into the bladder as a treatment for urological diseases. Because more than one siRNA can be encapsulated and targeted in the same nanoparticle, we can downregulate more than one urologic disease pathway with the same nanoparticles. Our first test of these nanoparticles will use a mouse model of bladder cancer. Thus, we can determine the therapeutic potential of siRNA for treatment of urologic diseases.